Reversible cardiac fibrosis and heart failure induced by conditional expression of an antisense mRNA of the mineralocorticoid receptor in cardiomyocytes

Abstract

Cardiac failure is a common feature in the evolution of cardiac disease. Among the determinants of cardiac failure, the renin-angiotensin-aldosterone system has a central role, and antagonism of the mineralocorticoid receptor (MR) has been proposed as a therapeutic strategy. In this study, we questioned the role of the MR, not of aldosterone, on heart function, using an inducible and cardiac-specific transgenic mouse model. We have generated a conditional knock-down model by expressing solely in the heart an antisense mRNA directed against the murine MR, a transcription factor with unknown targets in cardiomyocytes. Within 2-3 mo, mice developed severe heart failure and cardiac fibrosis in the absence of hypertension or chronic hyperaldosteronism. Moreover, cardiac failure and fibrosis were fully reversible when MR antisense mRNA expression was subsequently suppressed.

Figure 1

Inducible expression of MR-AS RNA allows cardiac specific and Dox-controlled down-expression of endogenous MR RNA expression. (a) To obtain cardiac-specific doxycycline-controlled transgene expression, a α-MHC tTA transgenic mouse line (transactivator mouse line), expressing the tTA transactivator under the control of the cardiac-specific α-MHC promoter, was mated with MR-AS transgenic mice (MR-AS mouse line), leading to double transgenic mice with inducible and cardiac-restricted expression of an antisense mRNA of the murine MR and LacZ as a reporter gene. (b) MR antisense mRNA (RNase protection assay) is expressed in 1-mo-old DT27 mice, whereas no expression is detectable in mono-transgenic littermates used as controls. Expression is shut down when DT mice received Dox for 10 d. (c) MR antisense mRNA expression resulted in a decrease of ≈50% of the cardiac endogenous MR mRNA in 1-mo-old DT27 (black bars; n = 5) as compared to controls (open bars; n = 4); 10-d Dox treatment restored MR mRNA levels (black bars: +Dox) close to control values (n = 4). By contrast, renal expression of MR was not modified. *, Control vs. DT mice and #, Dox-treated vs. untreated mice, when P < 0.05.

Figure 2

Expression of mMR-AS mRNA in the heart causes dilated cardiomyopathy. (a) Control mouse (4-mo-old) (Cont) and 4-mo-old DT27 mouse with decompensated heart failure (DT). (b) Survival of mice expressing the MR-AS mRNA in the heart. More than 70% of DT17 (■, n = 14) and ≈25% of DT27 (▴, n = 16) males died by 12 wk, whereas control littermates (○, n = 20) did not. None of the Dox-treated DT17 (□, n = 7) died within 12 wk. (c) Heart from a control mouse (Cont) and a DT27 mouse with a major cardiac hypertrophy (DT). (d) Heart/body weight ratio of 3-mo-old control mice (open bars) and DT27 mice (black bars), treated or not with Dox from day 8 postcoitum. In DT mice, MR antisense mRNA expression induces an increase in heart/body weight ratio, which could be prevented by turning down transgene expression with Dox treatment (n = 11 for each group).*, Control vs. DT mice and #, Dox-treated vs. untreated mice when P < 0.05. (e) Time course of cardiac phenotype in control or DT27 mice. Mice were analyzed at 4, 8, and 12 wk of age. Increase of heart/body weight ratio was observed in 2-mo-old DT mice (▴), whereas it was unchanged in control mice (▵) (n = 6–10 in each group; *, P < 0.05).

Figure 3

The cardiopathy induced by MR antisense mRNA expression is associated with cardiac remodeling and extensive interstitial fibrosis. (a) Histological analysis showed that DT heart (DT) is pathologic with large hyperchromatic nuclei (Inset), myocardial fiber disarray and myocyte hypertrophy that are absent in the control mouse (Left, cont) with (×1,000). (b) Sirius red-stained sections of myocardium of a DT (DT) and a control mouse (Cont). The interstitial fibrosis observed in DT is diffuse over the section (on RV as well as LV) (×200). (c) Interstitial cardiac fibrosis in 3-mo-old DT27 mice (black bars, n = 11), as compared to untreated mice (open bars, n = 11), is prevented when mice are treated with Dox from day 8 postcoitum. *, Control vs. DT mice and #, Dox-treated vs. untreated mice, when P < 0.05. (d) Quantification of cardiac fibrosis in control (▵) and DT27 (▴) mice reveals major interstitial fibrosis in 2- and 3-mo-old animals with a mild increase in interstitial fibrosis in 1-mo-old DT mice (n = 6–10 for each group). *, Control vs. DT mice and #, Dox-treated vs. untreated mice, when P < 0.05).

Figure 4

Spironolactone, an MR antagonist, has synergistic effects with MR-AS mRNA expression on cardiac phenotype. Control mice (open bars) or DT27 mice (black bars) were treated or not with 20 mg/kg/d spironolactone (spiro) for 8 wk after weaning. Spironolactone-treated DT mice (n = 9) showed an increase in the heart/body weight ratio (a) and the interstitial fibrosis (b), as compared to nontreated DT mice (n = 20). Spironolactone had no effect in control mice (n = 15), as compared to nontreated control mice (n = 20). *, Control vs. DT mice and #, spiro-treated vs. untreated mice, when P < 0.05.

Figure 5

The cardiopathy is reversible on Dox administration. (a) A 4-mo-old DT27 with decompensated heart failure (Right, DT, day 0) was treated with Dox. Rapid improvement of edema was observed. Within 10 d, the mouse recovered a weight similar to that of the control littermate (Left, control). (b and c) Dox administration to 2-mo-old DT mice (black bars) allowed improvement of the EF (b) and of the Vcfc (c) as early as 1 wk after Dox administration (open bars: control animals). Differences between treated and nontreated DT animals are significant after 3 wk of Dox administration (n = 5–7 in each group). #, Dox-treated vs. untreated DT mice, when P < 0.05. (d and e) A 1-mo Dox administration to 2-mo-old DT mice (8 wk + reversal, black bar) allows complete normalization of the heart/body weight ratio (d) and the reversal of the interstitial fibrosis (e) observed in nontreated DT mice (12 wk, black bar), to levels of control mice (open bars) (n = 7–10 in each group).*, Control vs. DT mice and #, Dox-treated vs. untreated mice, when P < 0.05.